Journal of Molecular Catalysis B-enzymatic最新文献

A fibrinolytic protease was purified from the culture supernatant of a GRAS fungus, Neurospora sitophila. The enzyme displayed a molecular mass of 34 kDa, as estimated by SDS-PAGE and gel filtration chromatography. The isoelectric point (pI) of the enzyme was 9.3 ± 0.2 as determined by iso-electric focusing (IEF). It was maximally active at pH 7.6 and 41 °C and displayed remarkable stability in a wide pH range (4–11) and up to 52 °C. The enzyme activity was inhibited by phenylmethane sulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA), indicating that it is a metal-dependent serine protease. It was found to be a direct acting plasmin like protein which efficiently cleaved the α-chain of fibrin(ogen), followed by β-chain and γ-chain. Three internal peptide sequences LASTANSGVLSGLLAGTVGGK; AYTSKSSVPSSVGLAR; LLDTGLNTAHSDFNR were determined by Q-TOF2. These results indicate no sequence similarities with other fibrinolytic enzymes suggesting it to be a novel enzyme. This fibrinolytic enzyme may be developed as a safe potential candidate for oral administration as a functional food additive or as a drug for prevention and/or treatment of thrombolytic diseases.

The thermal stabilization of enzymes is a critical factor in the development and reliability of enzyme-based processes and functional materials. Using a simple amine coupling approach for enzyme immobilization onto magnetic microbeads, followed by encasement of the beads in a hydrogel, we demonstrate that the thermal stability of the enzyme acetylcholinesterase can be increased dramatically. For example, when free and microbead-immobilized enzyme (“EM Conjugate”) are incubated overnight in a dry state at 63 °C (140 °F), the catalytic efficiency (k_cat/K_m) of the latter is higher than the former by six orders of magnitude (a factor of 2.16 × 10⁶). This effect arises mostly through a ∼29,700-fold decrease in K_m experienced by the EM Conjugate, relative to that of the free enzyme. Encapsulation of the EM Conjugate in a hydrogel based on poly(N-(3-aminopropyl methacrylamide)), which contains a primary amine, affords the enzyme additional stability when incubated overnight at 63 °C in an aqueous state. For example, its catalytic efficiency is four orders of magnitude higher than that of both the free enzyme (a factor of 4.34 × 10⁴) and that of the EM Conjugate alone (a factor of 1.78 × 10⁴) after all are incubated overnight at 63 °C. The presence of the hydrogel also caused the Michaelis constant to decrease by 1.38 × 10⁴ relative to that of the EM Conjugate, reaching a value of 2.18 × 10⁻³ M. Thus the hydrogel enables the AChE substrate binding site to retain a significant amount of its natural affinity for the substrate, after heating. This effect may occur via ion-pairing by the primary amines in the hydrogel polymer repeat unit, which are protonated and positively-charged at the assay pH. To the best of our knowledge, this simple method for enzyme thermal stabilization is novel and has not yet been investigated.

In this work, we explored the activation of a lipoprotein lipase from Burkholderia species by glucose-headed surfactants (GHSs) for enhancing its catalytic activity in organic solvent. Three GHSs were prepared and then tested as the additives for inducing the activation of lipoprotein lipase. The kinetic parameters of GHS-treated lipoprotein lipase were determined for the hydrolysis or alcoholysis of p-nitrophenyl acetate. It was found that GHS-treated lipoprotein lipase was 4 to 5 orders of magnitude more active than its native counterpart in organic solvent. Such a dramatic activity enhancement was largely the result of a huge increase in the turnover frequency k_cat. Surprisingly, the k_cat values in organic solvent were one order of magnitude greater than their aqueous counterparts. As a result, the k_cat/K_m of GHS-treated lipoprotein lipase in organic solvent became comparable to the aqueous level within one order of magnitude. We thus have demonstrated for the first time that a lipase can display nearly aqueous-like activity in organic solvent. As an illustrative application of GHS-treated lipoprotein lipase, we performed the dynamic kinetic resolution of two secondary alcohols, which provided the products of high enantiopurity (98–99%ee) with high yields (90–91%).

Lipase-based cross-linked aggregates were investigated for a non-specific reaction, i.e. the epoxidation of α-pinene to its oxygenated derivatives. The activity of the biocatalysts has been evaluated in a green context, i.e. ethyl acetate as both acetate-supplier and organic solvent with H₂O₂/UHP/TBHP as oxidant. Screening of the lipase sources indicated Aspergillus niger lipase as the most efficient biocatalyst for this reaction. Different immobilization protocols ((i) cross-linked enzyme aggregates (CLEA), (ii) cross-linked enzyme aggregates onto magnetic particles (CLEMPA) and (iii) covalent immobilized enzyme (CIE) onto magnetic particles (MP)) were evaluated considering the activity as main parameter. Thus, CLEA and CLEMPA afforded better epoxidation yields of α-pinene towards CIE. The investigated biocatalytic systems allowed to transform α-pinene into oxigenated derivatives with industrial and commercial applications (e.g. α-pinene oxide, camphene, pinanediol and camphonelic aldehyde). FTIR investigations on the biocatalysts revealed the effects of the immobilization protocol on the enzyme secondary-structure. Additionally, textural characterizations were performed by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) analysis.

Laccase-catalyzed synthesis of dye molecules represents a green choice to reduce the environmental footprint of conventional synthetic processes. Textile industry will benefit from this green technology since the synthesized dyes can be exploited to colour different fabrics.

This work describes the application of the Pleurotus ostreatus POXA1b laccase in polymeric dye synthesis using resorcinol and 2,5-diaminobenzenesulfonic acid (2,5-DABSA) as substrates. The potential of the resorcinol/2,5-DABSA coupling route was transferred to a chemical industry, Setaş Colour Center, by introducing a greener synthesis step within the process routinely used for textile dyeing. Dye synthesis was performed at different precursor ratios (1:1 and 1:10 2,5-DABSA: resorcinol) and their dyeing properties were compared on different fibres. The two mixtures of synthesized dyes proved to be effective on nylon and wool, with 1:10 ratio displaying the best performances in terms of dyeing efficiency and colour strength. Good and comparable end quality and “performances during use” were observed for nylon and wool coloured with both synthesized dyes.

The present work deals with the synthesis of an electrically conductive polycarbazole-titanium dioxide (PCz/TiO₂-6%) nanocomposite employing facile in-situ oxidative polymerization of carbazole monomer. In order to immobilize the yeast alcohol dehydrogenase (YADH) enzyme, the polymerization reaction was done in the presence of TiO₂ (titanium dioxide). The pristine PCz and PCz/TiO₂-6% nanocomposites were fully characterized using Fourier transform infra-red spectroscopy, Scanning electron microscopy, Transmission electron microscopy, Thermogravimetric analysis and Differential thermal analysis. The studies revealed that the TiO₂ and YADH loading changes nanocomposite morphology in comparison to pristine PCz. YADH immobilization was efficient and successfully carried out on PCz and PCz/TiO₂-6% nanocomposite with a loading efficiency of 67.4% and 88.2% respectively. Immobilized YADH on the PCz/TiO₂-6% nanocomposite enhanced YADH stability, recycling efficiency, and residual activity, which makes it ideally suited for industrial applications. A total of four 3D molecular field descriptors or field points were used to characterize and define the necessary properties required for a molecule to bind into a specified active site, in a characteristic fashion. 3D molecular dynamics and a molecular docking simulation were employed to predict the modes of interactions of YADH with either PCz or PCz/TiO₂-6%. The in vivo cytotoxicity profiles of PCz and PCz/TiO₂-6% nanocomposite were obtained by lethality bioassay against brine shrimp nauplii.

This paper describes the enzyme immobilization protocol as well as the enzymatic method for the direct resolution of (R,S)-atenolol. The used magnetic enzyme carriers possess on their surface new-synthetized chitosan derivatives with free amine groups distanced by ethyl or butyl chain. Additionally the catalytic activity of two types of commercially available lipases from Candida rugosa immobilized onto two different magnetic nanoparticles were compared. The highest values of enantioselectivity (E = 66.9), enantiomeric excess of product (ee_p = 94.1%) and conversion (c = 41.84%) were obtained by using lipase from Candida rugosa OF immobilized onto Fe₃O₄-CS-EtNH₂. The study confirmed that even after 5 reaction cycles the immobilized lipase maintain its high catalytic activity.